The differentiation of human embryonic stem (hES) cells to multiple lineages in culture offers unprecedented opportunities to investigate the earliest stages of human development and to generate large numbers of specific cell types for cell-based therapy. An essential step toward realizing this potential is the availability of an infrastructure that supports human ES cell research. Challenges include that the growth and differentiation of cells must become standardized to enable investigators from different disciplines to easily utilize this in vitro system. At the center of these efforts will be a Core facility that is designed to develop hES cell technologies and to support and train the research community at Mount Sinai. In addition,we will address fundamental questions relating to hES cell biology and hES cell-based technologies. To fully exploit the potential of hES cells, it is important to develop approaches allowing for the introduction of genes that can impact their differentiation as well as those that can prevent the rejection of their progeny, following transplantation into recipients. We will address these issues by developing approaches for site-specific genetic modification of the hES cells, through the use of adeno-associated viruses (AAV) and bacteriophage integrases, respectively. We will take advantage of the erythroid differentiation potential of hES cells as documented in the Preliminary Studies, to address questions relating to the mechanisms of the regulation of human globin gene expression. We will investigate the roles of FOXO transcription factors and oxidative stress on hES cell growth and erythroid specific differentiation. The outcome of these studies will provide a comparison of the growth and differentiation potential of multiple hES cell lines, new approaches for site-specific genetic modification of these cells, insights into the role of FOXO proteins on hES cell growth and differentiation and new information on the regulation of globin gene switching during the early stages of human erythroid development. We propose to use the following ES cells lines: ES01, ES02, ES03, ES04, ES05, ES06, T3-03, TE-04, TE-06, UC01, UC06, MI01, BG01, BG02, BG03, WA01, WA07, WA09, WA13, WA14.